The present invention relates to the preparation of compositions which comprise an antifungal compound of the polyene type such as natamycin, and the use of such compositions to treat foodstuffs or agricultural products or for pharmaceutical purposes.
For more than 20 years, natamycin has been used to prevent the growth of mould on cheese and sausages. (Ref. 1-12).
Cheeses are treated by immersion in a suspension of natamycin in water or by coating with a natamycinxe2x80x94containing aqueous emulsion of a polymer, commonly an aqueous emulsion of polyvinyl acetate. Sausages are mainly treated by immersion or by spray coating with a suspension of natamycin in water. Usually aqueous suspensions of natamycin for immersion treatments contain 0.1 to 0.2% w/v of natamycin, while polymer emulsions for coating purposes containing natamycin have a natamycin content of 0.01 to 0.05% w/v.
Mostly these treatments are highly effective in preventing spoilage by mould. However, because of the low solubility of natamycin, spoilage by some fungi which have relatively low susceptibility to natamycin may still occur due to lack of full inhibition.
The present invention relates to a process for improving the activity of polyene antifungal compounds, in particular natamycin, towards mould, and to compositions prepared according to said process. In particular, such compositions are useful for combatting yeasts and fungi which are normally relatively resistant to the action of antifungal compounds of the polyene type.
Unexpectedly, it has been found that the activity of antifungal compounds of the polyene type, such as natamycin, towards yeast and fungi can be markedly enhanced by one of the following methods.
(1) By modifying the polymorphic form. Firstly the polyene antifungal compound can be converted into a solvated form by contacting the compound with a solvent. An example of such a solvate is natamycin methanol solvate.
Secondly the solvated solvent molecules may be removed from the crystal lattice of the solvate. An example of such a compound is xcex4-natamycin, which can be obtained by subjecting natamycin methanol solvate to diminished pressure over phosphorous pentoxide.
Thirdly the xe2x80x9cemptyxe2x80x9d room of the crystal lattice may be refilled with other solvent molecules; e.g. by contacting the xe2x80x9cemptyxe2x80x9d compound with a solvent or the vapour thereof. Examples of such compounds are the solvates of natamycin with the following solvents; methanol, ethanol, n-propanol, n-butanol, methoxy-ethanol, dichloromethane, 1,2-dichloro-ethane, methyl formate, ethyl formate, acetonitrile, formamide, N-N-dimethyl formamide and dimethyl sulphoxide.
Finally new polymorphic forms of polyene antifungal compounds may be obtained by subjecting the compound with the xe2x80x9cemptyxe2x80x9d crystal lattice to a humid condition. For example when xcex4-natamycin is subjected to 76% RH a new trihydrated crystal modification is obtained. This new compound, which is named xcex3-natamycin, has an enhanced activity towards species which are normally relatively resistant to the action of the original xcex1-natamycin, which is also a trihydrated form of natamycin (see Ref. 14, p. 520).
(2) Prior to incorporating the antifungal compound into a food treatment composition or into a pharmaceutical preparation the antifungal compound is dissolved in a suitable solvent system.
Suitable solvent systems for dissolving the fungicide are lower alcohols, e.g. a lower alcohol having 1 to 4 carbon atoms, glacial acetic acid, aqueous acid and alkaline solutions, and appropriate mixtures thereof. Examples of suitable alcohols are methanol, ethanol, n-propanol, isopropanol, ethylene glycol, ethoxy ethanol, propylene glycol and glycerol. Examples of suitable acidic solutions are aqueous solutions of hydrochloric acid, citric acid and acetic acid. Examples of suitable aqueous alkaline solutions are aqueous solutions of ammonia, alkali metal hydroxides and ethanolamine.
(3) By preparing an earthalkaline salt. Examples are the barium and the calcium salt of natamycin.
(4) By applying on a carrier. An example is natamycin on fumed silica, which can be prepared by mixing a solution of natamycin in methanol with fumed silica and removing the solvent by evaporating at diminished pressure. Preparations and compositions according to the invention are characterized by a high release profile of the antifungal compound in comparison to the unmodified polyene when measured by the method described in Example 1.
Therefore the invention relates to a method of preparing an anti-fungal composition containing an anti-fungal compound of the polyene type characterised in that said anti-fungal compound is incorporated into the composition in a modified form which shows an enhanced release profile of said anti-fungal compound in comparison to the unmodified anti-fungal compound. The anti-fungal compound can be brought in the modified form for example by modifying the polymorphic form and/or bringing the compound in a suitable solution. By unmodified form is meant the form given in xe2x80x98The Merck Indexxe2x80x99, an encyclopedia of chemicals, drugs and biologicals or by the production referred to in The Merck Index.
According to one aspect of the invention a novel natamycin form and/or compound is disclosed. This compound is preferably selected from the group consisting of
Natamycin methanol solvate, stabilized by storing under nitrogen gas and/or at xe2x88x9230xc2x0 C.;
xcex4-natamycin;
xcex3-natamycin;
Calcium salt of natamycin;
Barium salt of natamycin;
The solvates of natamycin with: ethanol: 1-propanol, 1-butanol, 2--methoxyethanol, methyl formate, ethyl formate, acetone, dichloromethane, acetonitrile dimethylformamide, dimethyl sulphoxide, and formamide; and
Natamycin on fumed silica as a carrier.
These compounds all have an improved release of the antifungal compound compared to unmodified xcex1-natamycin.
Thus the present invention also provides a method of assessing the efficacy of an anti-fungal composition for inhibition of fungal or yeast cell growth at a surface which comprises the steps of:
(i) applying a sample of the composition to a carrier;
(ii) applying the carrier thus loaded to the surface of an agar plate containing yeast or fungal cells such that the composition is in contact with the agar surface;
(iii) incubating the agar plate and carrier at a temperature such that growth of said cells is inhibited; and
(iv) removing the carrier from the agar plate;
(v) incubating the agar plate under growth permitting conditions for said cells; and
(vi) determining inhibition of cell growth in the agar due to the presence of fungicide released from the composition sample.
In particular, the method is suitable for determining the availability of active anti-fungal components from preparations meant for surface treatments. Further the method is especially suitable for the determination of the dissolution of active anti-fungal components which have a low solubility in water.
Preparations containing activated fungicide are useful for preventing the growth of fungi which are normally relatively resistant to the action of polyene fungicides. Examples of fungi which have relatively low susceptibility towards natamycin are Verticillium cinnabarinum, Botrytis cinerea and Trichophyton species. Among Aspergillus, Fusarium and Penicillin species, species exhibiting relatively high tolerance to natamycin may also be found. An example of such a species is Penicillium echinulatum var. discolor. 
Thus, in one aspect the present invention provides methods of preparing an anti-fungal composition containing an anti-fungal compound of the polyene type which is activated by one of the above mentioned methods. In a further aspect, the invention also provides compositions thus prepared.
In yet another aspect, the invention provides a method of treating a food or agricultural product to inhibit yeast or fungal growth which comprises applying a composition of the invention.
In principle, a solution of an anti-fungal compound of the polyene type may be incorporated into all kinds of preparations which are suitable for treating foodstuffs such as cheese and sausages and/or for treating agricultural products such as fruit, vegetables and seed. Suitable preparations for treating foodstuffs such as cheese and sausages are, for instance, coating emulsions which can be applied by brushing or by using a spray device and liquid preparations which can be used for immersion treatments. Preferably the pH of a preparation according to the invention for food or agricultural product treatment is from 1 to 10, more preferably from 2 to 8 and most preferably from 3.5 to 7.5. Further, the invention concerns foods and agricultural products when so treated.
Finally, the activated fungicide of the polyene type may also be incorporated into pharmaceutical preparations, for instance for topical application. Examples of suitable pharmaceutical preparations are lotions, creams and ointments.